Postoperative pain is a major morbidity, and persistent opioid use after surgery has contributed to an epidemic. An improved mechanistic understanding of how pain is regulated within the brain can lead to novel non-opioid analgesic development. The long-term goal of this proposal is to understand the central regulation of postoperative pain. The objective of the current application is to define the role of prelimbic cortex (PL) and anterior cingulate cortex (ACC), two key components of the prefrontal cortex in rodents, in the regulation of acute and chronic postoperative pain. The PL is homologous to human dorsolateral prefrontal cortex that is known to undergo synaptic changes with chronic pain, and the ACC is a well-described region for processing affective component of pain across species. Our central hypothesis is that an imbalance in neural activities in the PL and ACC contributes to symptoms of postoperative pain and thus forms a therapeutic target. Our hypothesis is supported by the current literature showing that the PL has a pain-inhibitory role, whereas the ACC enhances pain aversion, and that chronic pain causes increased excitability in the ACC and hypo-excitability in the PL. It is also supported by our recent results demonstrating that AMPAkines and ketamine, drugs that alter glutamate signaling and shape cortical circuits, reduce pain. In Aim 1, we will test the hypothesis that an imbalance in ACC and PL activities contributes to postoperative pain in awake freely behaving rats. We will use paw incision (PI) to mimic acute reversible incisional pain, and spared nerve injury (SNI) to model chronic pain after intraoperative nerve damage. We will first correlate imbalanced prefrontal activities with pain, by showing a concurrent loss of nociceptive response in the PL and gain of response in the ACC as pain behavior persists, and the resolution of such neural changes as pain resolves, using simultaneous in vivo extracellular recordings of the PL and ACC. Further, to test the causal effect of this imbalance on pain, we will show that optogenetic PL activation, or ACC inhibition, reverses postoperative pain behaviors. Next, we will use optrode recordings to dissect a local pain- regulatory circuit fromthe PL to the ACC. Further, we will use an unbiased supervised machine learning analysis to validate the relationship between the imbalance in PL and ACC activities and the chronicity of postoperative pain. In Aim 2, We will test the hypothesis that pharmacologic and electrical neuromodulation can target imbalanced PL/ACC activities in the postoperative pain state. We will show that AMPAkines and ketamine increase PL outputs and reduce ACC activities to inhibit pain and optimize the timing and dosing regimens for these drugs and test therapeutic synergy. We will also optimize invasive and non-invasive electrical stimulation protocols in the PL to treat pain. This project is innovative because it applies a new systems neuroscience approach with cutting-edge techniques to uncover a central pain-regulatory mechanism. The work is significant because it produces novel applications of FDA-approved drugs (ketamine and APMAkines) for postoperative pain and a blueprint for new deep brain or transcranial stimulation methods to treat pain.